Microbial GrowthMicrobial GrowthMicrobial Growth
ByBy
Dr. Carmen RexachDr. Carmen Rexach
MicrobiologyMicrobiology
Mt San Antonio CollegeMt San Antonio College
Microbial growth• Definition
– Increase in the number of cells– Increase in microbial mass
• Requires specific physical and chemical conditions
Classifications by temperature
• Psychrophiles• Psychrotrophs• Mesophiles• Thermophiles• Extreme thermophiles
Cold temperature
• Psychrophiles– [-10 to -18oC (optimum = -15)– Example: ocean floor
• Psychrotrophs– 0 to 30oC (optimum = 20)– Example: grow in refrigerator – freezing stops microbial growth,
does not necessarily kill bacteria
Moderate temperature
• Mesophiles– 10-48oC (optimum 25-40)– Examples: human pathogens,
spoilage, disease organism
Hot temperature
• Thermophiles– 40-72oC (optimum 50-60)– Example: hot springs
• Extreme thermophiles– 65-110oC – Example: hydrothermal vents– Taq polymerase = enzymes from Thermus aquaticus
pH
• Most organisms require 6.5-7.5 for optimum growth
• Acidophils– Bacteria tolerant to acidity
• Optimum pH in lab– Maintained by adding buffers (peptones,
phosphate salts, etc)
Osmotic pressure• Bacteria 80-90% water• Hypertonic solutions
– crenation• Hypotonic solutions
– Lysis of membrane– Prevented by cell wall
• Halophiles– Require salt for growth (Dead Sea)
• Facultative halophiles– Can grow in salt, not required
Carbon• All organisms require carbon source
– By definition: all organic molecules contain carbon and hydrogen
• Chemoheterotrophs– Utilize organic carbon sources
• Chemoautotrophs and photoautotrophs– Utilize inorganic carbon = CO2
Nitrogen, sulfur, phosphorus• N
– amino acid formation– Some bacteria fix inorganic nitrogen– Others use organic nitrogen = symbiosis
• S– amino acid synthesis and some vitamins
• P– synthesis of nucleic acids and
phospholipids
Trace elements
Small amount of mineral elementsUsually needed by enzymes as cofactorsInclude
FeCa++
Mg++
Oxygen
• All organisms using oxygen require special enzymes to protect cells from toxic effects of oxygen– Superoxide dismutase– Catalase or peroxidase
• Two categories based on oxygen requirements– Aerobes– Anaerobes
Aerobes• Aerobes = use oxygen• obligate aerobes = require oxygen• How much?
– Microaerophiles = grow in oxygen concentrations lower than air
– Facultative anaerobes = aerobes with the ability to grow in absence of oxygen• Intestinal bacteria, yeasts
Anaerobes
• Aerotolerant anaerobes– Tolerate oxygen– Can’t use it for growth– Many ferment carbohydrates to lactic acid
• Ex: Lactobacillus
• Obligate anaerobes– Killed by oxygen– Do not contain enzymes necessary to detoxify
Culture mediumAny material prepared for growth of bacteria in a labCulture = microbes growing in or on culture mediumAgar used to solidify culture
Types of culture media(start with sterile media)
• Chemically defined• Complex media• Anaerobic growth media/methods• Special techniques• Selective and differential media• Enrichment culture• Pure culture
Culture media• Chemically defined
– Know exact chemical composition• Complex
– Several components– Varies from batch to batch– Ex) nutrient agar, nutrient broth
• Special techniques– Ex) culturing certain bacteria or parasites on
living tissues or cells
Anaerobic growth media and methods
• Special reducing media– Thioglycolate broth– Ties up oxygen
• BBL jars– Removes oxygen chemically– Contains anaerobic indicator (methylene
blue)– Other options: anaerobic glove box
Culture media
• Selective and differential media– Encourages growth of selected microorganisms
• Ex) sabdex– Helps to identify certain organisms
• Ex) CCFA• Blood agar = α, β, γ hemolysis
• Enrichment culture– Mixed culture and want to encourage
preferential growth of one organism
Pure culture
• Techniques work on basis that each colony arises from division of single bacterial cell
• Two techniques– Pour plate– Streak plate
– Goal: to grow isolated colonies
Bacterial division
• Binary fission• Budding• Spore formation
– Actinomycetes (at tip of filaments)• Fragmentation
– Filamentous bacteria
Binary fission
Bacterial cell elongates. Bacterial DNA replicates.
Cell wall and plasma membrane grow inward from both sides, forming a septum. Cross wall forms and two cells are produced identical to the parent cell.
Generation time
• Amount of time it takes for cell to divide or for population to double
• Growth rate– Population growth = change in the number of
cells or cell mass per unit of time• Bacterial cells multiply logarithmically
– Rate varies among different organisms and under different nutritional and genetic influences
Lag phase
• Getting used to new environment– Lots of metabolism
• No lag phase – if inoculate from exponentially growing culture
• Lag phase – If inoculate from stationary phase due to
depletion of certain nutrients– If move from richer culture to poorer culture
Log phase & stationary phase
• Log phase– Exponential growth– Greatest metabolic activity– Increased susceptibility to adverse conditions
• Stationary phase– Number of microbial deaths equal to new cells
produced– Nutrient depletion, increased wastes, changes
in pH
Death or logarithmic decline
• Deaths exceed new cell formation• Exponential function but slower than
log phase
How to measure microbial growth• Plate counts
– Serial dilutions to keep number of colonies to be counted low
• Other methods– Direct count under microscope using slides with
counting chambers– Electronic cell counters, etc.– Indirect methods
• Turbidity = indicator of increased number• Measure amount of metabolic activity
– Amount of CO2 produced– Dessicate and weigh